Philipp Diehl

BMPER Is Upregulated by Statins and Modulates Endothelial Inflammation by Intercellular Adhesion Molecule-1

Objective—In addition to lowering cholesterol, statins exert pleiotropic effects on endothelial cells. Bone morphogenetic proteins (BMPs) have recently been implicated in vascular inflammation and disease. We set out to investigate the effect of statins on BMP endothelial cell precursor–derived regulator (BMPER), a novel member of the BMP pathway. Methods and Results—Mevastatin enhanced BMPER expression in cultured endothelial cells in a time- and concentration-dependent manner as determined by immunocytochemistry, RT-PCR, and Western blotting. Similar effects were observed in vitro and in vivo using simvastatin. Actinomycin D chase analysis and BMPER promoter reporter assays revealed that this is mostly a posttranscriptional event resulting in prolonged BMPER RNA half-life. We confirmed that the RhoA/Rho-associated coiled-coil containing protein kinase Rho kinase (Rock)/actin pathway is involved using the specific pathway activator cytotoxic necrotizing factor of Yersinia pseudotuberculosis, which prevented upregulation of BMPER expression by mevastatin and pathway inhibitors (C3-toxin, RhoAN19 mutant, fasudil, and cytochalasin D) that enhanced BMPER expression. Increasing concentrations of BMPER exert antiinflammatory features in endothelial cells as reflected by intercellular adhesion molecule–1 downregulation. Accordingly, silencing of BMPER enhances intercellular adhesion molecule–1 expression. Furthermore, mevastatin reduced the expression of proinflammatory BMP4, a well-known direct interaction partner of BMPER. Conclusion—Mevastatin modulates the BMP pathway by enhancing BMPER via the RhoA/Rock/actin pathway, as well as by reducing BMP4 expression. BMP4 downregulation and BMPER upregulation contribute to the antiinflammatory pleiotropic effects of statins.

Krüppel-like factor 15 regulates BMPER in endothelial cells

AIMSnBone morphogenetic proteins (BMPs) are involved in embryonic and adult blood vessel formation in health and disease. Previous studies have shown that BMP endothelial cell precursor-derived regulator (BMPER) plays an important role in endothelial cell function and blood vessel formation. BMPER is a key regulator of BMP4 activity and a prerequisite for BMP pathway activation by BMP4 in endothelial cells. Here, we characterize the BMPER promoter and elucidate mechanisms of BMPER regulation.nnnMETHODS AND RESULTSnTo investigate transcriptional mechanisms of BMPER expression, the murine BMPER promoter was cloned and characterized. A series of 5 deletions of the BMPER promoter revealed that the proximal promoter contains activating cis-elements. By overexpression or siRNA-based knockdown, we demonstrate that BMPER expression is activated by Krüppel-like factor (KLF) 15. As determined by gelshift analyses, KLF15 binds directly to a predicted KLF-binding element at -284 bp within the BMPER promoter. Co-expression experiments show that Sp1 acts as an antagonist for KLF15-induced promoter activation. Endothelin-1 was identified as a potent inhibitor of KLF15 and BMPER expression in endothelial cells, suggesting that KLF15 is a transducer of endothelin-1 activity on BMPER expression. The selective ET(B) endothelin receptor antagonist BQ788 abolished the downregulation of BMPER expression by endothelin-1.nnnCONCLUSIONnMechanistically, we found that KLF15 is a strong and direct activator of the BMPER expression. BMPER is downregulated by endothelin-1 in a dose-dependent fashion and in parallel to KLF15. As KLF15 deficiency is accompanied by a vascular phenotype and BMPER is necessary for proper blood vessel formation, we suggest a chain of events in which the effects of endothelin-1 on BMPER are mediated by KLF15.

Editorial Comment: Reduction of oxidative stress: a new indication for acetylsalicylic acid in coronary artery bypass surgery

Thus, ASA reduces systemic inflammation in CABG patients without increasing the risk of bleeding complications. Although patient numbers are small in the present study, the results are in accordance with recent subanalyses of large randomized trials on third generation adenosine diphosphate (ADP)-receptor antagonists. In these trials, evaluating prasugrel or ticagrelor compared with clopidogrel—all in combination with ASA— patients receiving the stronger ADP-receptor antagonists fared better in terms of mortality even though bleeding was increased [9, 10]. Thus, treatment with antiplatelet drugs is beneficial not only in patients with acute coronary syndromes or coronary stent implantation, but also in patients undergoing CABG. Many surgeons understandably fear the increased bleeding risk that comes along with potent antiplatelet drugs. This risk has not been substantiated in the present study, a finding that may be partially due to the small sample size. However, even if patients did bleed more when treated with ASA or other potent antiplatelet drugs in the perioperative phase, is this not to be accepted in view of the overall benefit and improved survival? Patients will be grateful if their surgeon is willing to be extra careful and to deal with perioperative bleeding in order to allow them to take advantage of these drugs. Obviously, it remains to be elucidated by future research that the mechanism of ASA contributes more to better outcomes—antioxidative features or platelet inhibition. However, Berg et al. ought to be congratulated for having stimulated this discussion by their intriguing findings. Independent of the underlying mechanism, patients planning to undergo CABG should receive ASA whenever possible.

Bone Morphogenetic Protein-Modulator BMPER Regulates Endothelial Barrier Function

The endothelium serves as a selective barrier and controls the exchange of nutrients, hormones, and leukocytes between blood and tissues. Molecular mechanisms contributing to the pathogenesis of endothelial barrier dysfunction remain incompletely understood. Accumulating evidence implicates bone morphogenetic protein (BMP)-modulator BMPER as a key regulator in endothelial biology. Herein, we analyze the impact of BMPER in the control of endothelial barrier function. To assess the role of BMPER in vascular barrier function in mice, we measured the leakage of Evans blue dye from blood into interstitial lung tissue. BMPER+/− mice exhibited a significantly higher degree of vascular leak compared with wild-type siblings. In accordance with our in vivo observation, siRNA-based BMPER knockdown in human umbilical endothelial cells increased endothelial permeability measured by FITC-dextran passage in transwell assays. Mechanistically, BMPER knockdown reduced the expression of VE-cadherin, a pivotal component of endothelial adherens junctions. Conversely, recombinant human BMPER protein upregulated VE-cadherin protein levels and improved endothelial barrier function in transwell assays. The effects of BMPER knockdown on VE-cadherin expression and endothelial permeability were induced by enhanced BMP activity. Supporting this notion, activation of BMP4-Smad-Id1 signaling reduced VE-cadherin levels and impaired endothelial barrier function in vitro. In vivo, Evans blue dye accumulation was higher in the lungs of BMP4-treated C57BL/6 mice compared to controls indicating that BMP4 increased vascular permeability. High levels of BMPER antagonized BMP4-Smad5-Id1 signaling and prevented BMP4-induced downregulation of VE-cadherin and endothelial leakage, suggesting that BMPER exerts anti-BMP effects and restores endothelial barrier function. Taken together, this data demonstrates that BMPER-modulated BMP pathway activity regulates VE-cadherin expression and vascular barrier function.

Coronary magnetic resonance imaging after routine implantation of bioresorbable vascular scaffolds allows non-invasive evaluation of vascular patency

Background Evaluation of recurrent angina after percutaneous coronary interventions is challenging. Since bioresorbable vascular scaffolds (BVS) cause no artefacts in magnetic resonance imaging (MRI) due to their polylactate-based backbone, evaluation of vascular patency by MRI might allow for non-invasive assessment and triage of patients with suspected BVS failure. Methods Patients with polylactate-based ABSORB-BVS in proximal coronary segments were examined with 3 Tesla MRI directly (baseline) and one year after implantation. For assessment of coronary patency, a high-resolution 3D spoiled gradient echo pulse sequence with fat-saturation, T2-preparation (TE: 40 ms), respiratory and end-diastolic cardiac gating, and a spatial resolution of (1.08 mm)3 was positioned parallel to the course of the vessel for bright blood imaging. In addition, a 3D navigator-gated T2-weighted variable flip angle turbo spin echo (TSE) sequence with dual-inversion recovery black-blood preparation and elliptical k-space coverage was applied with a voxel size of (1.14 mm)3. For quantitative evaluation lumen diameters of the scaffolded areas were measured in reformatted bright and black blood MR angiography data. Results 11 patients with implantation of 16 BVS in the proximal coronary segments were included, of which none suffered from major adverse cardiac events during the one year follow up. Vascular patency in all segments implanted with BVS could be reliably assessed by MRI at baseline and after one year, whereas segments with metal stents could not be evaluated due to artefacts. Luminal diameter within the BVS remained constant during the one year period. One patient with atypical angina after BVS implantation was noninvasively evaluated showing a patent vessel, also confirmed by coronary angiography. Conclusions Coronary MRI allows contrast-agent free and non-invasive assessment of vascular patency after ABSORB-BVS implantation. This approach might be supportive in the triage and improvement of diagnostic workflows in patients with postinterventional angina and scaffold implantation. Trial registration German Register of Clinical Studies DRKS00007456

A ligand-specific blockade of the integrin Mac-1 selectively targets pathologic inflammation while maintaining protective host-defense

Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.Integrin-based therapeutics could block inflammatory processes but they also impair host defence, limiting their usefulness. Here the authors report an anti-Mac1 antibody that blocks its interaction with pro-inflammatory ligand CD40L but not other ligands, and show that it can protect against sepsis in mice.

The effect of oxygen in Sirt3-mediated myocardial protection: a proof-of-concept study in cultured cardiomyoblasts

Sirtuin 3 is a nicotinamide adenine dinucleotide dependent mitochondrial deacetylase that governs mitochondrial metabolism and oxidative defense. The demise in myocardial function following myocardial ischemia has been associated with mitochondrial dysfunction. Sirt3 maintains myocardial contractile function and protects from cardiac hypertrophy. The role of Sirt3 in ischemia is controversial. Our objective was to understand, under what circumstances Sirt3 is protective in different facets of ischemia, using an in vitro proof-of-concept approach based on simulated ischemia in cultured cardiomyoblasts. Cultured H9c2 cardiomyoblasts were subjected to hypoxia and/or serum deprivation, the combination of which we refer to as simulated ischemia. Apoptosis, as assessed by Annexin V staining in life-cell imaging and propidium-iodide inclusion in flow cytometry, was enhanced following simulated ischemia. Interestingly, serum deprivation was a stronger trigger of apoptosis than hypoxia. Knockdown of Sirt3 further increased apoptosis upon serum deprivation, whereas no such effect occurred upon additional hypoxia. Similarly, only upon serum deprivation but not upon simulated ischemia, silencing of Sirt3 led to a deterioration of mitochondrial function in extracellular flux analysis. In the absence of oxygen these Sirt3-dependent effects were abolished. These data indicate, that Sirt3-mediated myocardial protection is oxygen-dependent. Thus, mitochondrial respiration takes center-stage in Sirt3-dependent prevention of stress-induced myocardial damage.

Moderate ischemic mitral regurgitation. Coronary artery bypass grafting with versus without simultaneous treatment of the mitral valve is associated with comparable long-term survival.

BACKGROUNDnIschemic mitral regurgitation (IMR) is a frequent complication of coronary artery heart disease and is associated with increased mortality. Controversies exists whether patients with moderate IMR may benefit from a combined procedure with coronary artery bypass grafting (CABG) and treatment of mitral regurgitation.nnnMETHODSn451 patients with moderate IMR (grade 2) receiving either CABG alone (CABG only) or additional mitral valve repair or replacement (CABG+MV) were included in this observational single-centre study. Patients were matched according to the number of bypass grafts, preoperative NYHA functional class and age. In total, 42 patients (21 CABG only, 21 CABG+MV) were analysed.nnnRESULTSnThe mean follow-up was 4.3±1.9 years. CABG alone reduced the IMR grade to 1.2±0.5, whereas the combined procedure resulted in a decrease to 0.6±0.7 (P=0.039). NYHA functional class was reduced from grade 2.8±0.6 to 1.6±0.5 (CABG only) and 2.0±1.1 (CABG+MV, P=0.55). Three- and five-year survival rates were 71% and 53% in the CABG only group compared to 60% both after 3 and 5 years in the CABG+MV group (P=0.89).nnnCONCLUSIONSnIn this observational study, combined CABG and surgical treatment of moderate IMR was associated with a more effective reduction of IMR than CABG alone. No statistically significant differences in survival or NYHA functional class were observed. Further studies will need to investigate the value of additional mitral valve surgery in moderate IMR.

Endothelial BMP4 Regulates Leukocyte Diapedesis and Promotes Inflammation

Leukocyte recruitment is a fundamental event in the response of the innate immune system to injury. This process is promoted in part by the opening of endothelial cell adherens junctions that allows leukocyte extravasation through gaps between adjacent endothelial cells. VE-cadherin is a key component of endothelial cell adherens junctions and a negative regulator of leukocyte emigration. Accumulating evidence implicates bone morphogenetic protein (BMP) 4 as a critical regulator in vascular biology, but its role in leukocyte extravasation in vitro and in vivo has not been investigated so far. To assess the impact of BMP4 on leukocyte emigration in vivo, we used the thioglycollate-induced peritonitis model. C57BL/6 mice were intraperitoneally (i.p.) injected with recombinant BMP4 in addition to thioglycollate. Compared to solvent-treated controls, we observed higher accumulation of leukocytes in the peritoneal lavage of BMP4-treated mice indicating that BMP4 promotes leukocyte diapedesis into the inflamed peritoneal cavity. Endothelial cell-specific deletion of BMP4 in mice markedly diminished leukocyte diapedesis following thioglycollate administration suggesting that endothelial BMP4 is required for leukocyte recruitment. Consistent with these in vivo results, transwell migration assays with human umbilical vein endothelial cells (HUVECs) in vitro revealed that recombinant BMP4 enhanced leukocyte transmigration through the endothelial monolayer. Conversely, silencing of endothelial BMP4 by siRNA dampened leukocyte diapedesis in vitro. Mechanistic studies showed that loss of BMP4 improved endothelial junction stability by upregulation of VE-cadherin expression in vitro and in vivo. Vice versa, treatment of HUVECs with recombinant BMP4 decreased expression of VE-cadherin and impaired endothelial junction stability shown by Western blotting and immunocytochemistry. Finally, severe endothelial damage in HUVECs in response to serum of patients collected 24xa0h after survived cardiac arrest was accompanied by increase in leukocyte migration in transwell assays and activation of the BMP pathway most probably by upregulation of endothelial BMP4 RNA and protein expression. Collectively, the present study provides novel evidence that endothelial BMP4 controls leukocyte recruitment through a VE-cadherin-dependent mechanism and that BMP4-induced inflammation might be involved in the pathogenesis of endothelial cell damage following successful resuscitation after cardiac arrest.

Antiplatelet therapy after acute coronary syndrome. Therapeutic strategies and treatment duration

Besides percutaneous coronary interventions, antiplatelet drugs are of overwhelming importance for patients with acute coronary syndrome (ACS). For ACS patients, the guidelines recommend treatment with acetylsalicylic acid and a P2Y12 receptor antagonist. The third generation P2Y12 receptor antagonists prasugrel and ticagrelor provide stronger platelet inhibition than clopidogrel and improve the clinical outcome in patients with ACS; however, it is still under discussion which P2Y12 antagonist fits best to which subgroup of ACS patients. This article summarizes current guidelines and antiplatelet treatment strategies for patients with non-ST-segment elevation (NSTE) ACS or ST-segment elevation myocardial infarction (STEMI). The information is mainly based on the recently published guidelines of the European Society of Cardiology on myocardial revascularization.ZusammenfassungNeben der perkutanen Koronarintervention (PCI) bildet die antithrombozytäre Therapie einen wesentlichen Bestandteil der Behandlung eines akuten Koronarsyndroms (ACS). Prinzipiell empfehlen Leitlinien, dass Patienten mit ACS ohne ST-Hebung (NSTE-ACS) oder ST-Hebungs-Infarkt (STEMI) eine Kombination aus Azetylsalizylsäure (ASS) und einem P2Y12-Antagonisten erhalten sollten. Orale P2Y12-Antagonisten der dritten Generation (Prasugrel und Ticagrelor) zeigen eine effektivere Thrombozytenaggregationshemmung im Vergleich zu Clopidogrel. Auch die klinische Überlegenheit dieser Substanzen konnte bereits in großen, randomisierten, kontrollierten Studien an Patienten mit ACS gezeigt werden. Welcher P2Y12-Antagonist die beste Wahl für welche Patientengruppe ist, wird kontrovers diskutiert. Dieser Artikel hat zum Ziel, eine Übersicht der Möglichkeiten der antithrombozytären Therapie bei Patienten mit NSTE-ACS oder STEMI zu geben. Die Leitlinien zur antithrombotischen Therapie wurden von der European Society of Cardiology (ESC) vor wenigen Wochen neu formuliert. Diese dienen als Grundlage der hier dargestellten Übersicht.AbstractBesides percutaneous coronary interventions, antiplatelet drugs are of overwhelming importance for patients with acute coronary syndrome (ACS). For ACS patients, the guidelines recommend treatment with acetylsalicylic acid and a P2Y12 receptor antagonist. The third generation P2Y12 receptor antagonists prasugrel and ticagrelor provide stronger platelet inhibition than clopidogrel and improve the clinical outcome in patients with ACS; however, it is still under discussion which P2Y12 antagonist fits best to which subgroup of ACS patients. This article summarizes current guidelines and antiplatelet treatment strategies for patients with non-ST-segment elevation (NSTE) ACS or ST-segment elevation myocardial infarction (STEMI). The information is mainly based on the recently published guidelines of the European Society of Cardiology on myocardial revascularization.